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PhD Thesis: Xiaobin Liu

Dissertation Abstract:
Models to Study Physiological and Behavioral Functions of the Neuropeptide S System

By Xiaobin Liu
Doctor of Philosophy in Pharmacology and Toxicology
University of California, Irvine, 2010
Professor Rainer K. Reinscheid, Chair

The goal of the studies presented in this dissertation is to advance our knowledge about molecular and anatomical features, as well as physiological and behavioral functions of the Neuropeptide S (NPS) system. NPS and its cognate receptor NPSR constitute a recently discovered ligand/receptor pair in the mammalian brain, but our understanding of its physiological roles is incomplete.

Studies presented in Chapter 2 describe the generation of a transgenic mouse model in which NPS-producing neurons co-express the fluorescent marker enhanced green-fluorescent protein (EGFP). NPS/EGFP-transgenic mice were used to determine the precise anatomical location of NPS-producing neurons in the mouse brainstem relative to neuronal markers, such as tyrosine hydroxylase. NPS/EGFP-positive neurons were detected in only two brainstem structures: the peri-locus coeruleus (periLC) and the Kölliker-Fuse (KF) nucleus.

Fluorescent cells from both nuclei were then harvested separately by laser-capture microdissection and RNA was extracted, converted to cDNA, and used to hybridize Affymetrix Mouse Genome expression microarrays in order to obtain a comprehensive gene expression profile. Expressed genes were specifically analyzed for co-expressed neurotransmitters, especially neuropeptides, and receptors.

Analysis of co-expressed neuropeptides by immunofluorescent staining of brain sections revealed that NPS-producing neurons of the KF nucleus also contain the neuropeptide galanin. Expression of a large number of GPCRs and ligand-gated ion channels was predicted from the microarray analysis and functional electrophysiological studies to verify their presence is part of an ongoing collaboration with Dr. Pape’s group at the Westfaelische-Wilhelms University in Muenster, Germany. Neuronal activation as a consequence of stress exposure was detected in NPS/EGFP-positive neurons by staining for the immediate-early gene marker c-fos. These data confirm earlier hypothesis that the NPS system might be part of the neuronal stress response network. NPS/EGFP-transgenic mice will be an invaluable model to study transmitter systems controlling NPS release and transmitters that are co-released together with NPS that might thus influence downstream effects caused by activation of NPS-producing neurons.

Studies presented in Chapter 3 describe the generation and phenotypical analyses of NPS precursor knockout mice. Exon 3 of the NPS precursor was deleted by targeted recombination in ES cells and several lines of knockout mice were obtained. Analyses of behavioral phenotypes revealed that NPS precursor knockout mice showed elevated anxiety-like behaviors, reduced exploratory activity and significantly attenuated long-term memory in an aversive memory paradigm. These observations confirm and extend previous studies that described anxiolytic, arousal-enhancing and procognitive effects of centrally administered NPS.